This invention relates to fluorescent 7-hydroxy coumarin compounds with substitutions in the 4 position having a length greater than one carbon atom. The compounds thus are derivatives of 4-methylumbelliferone (7-hydroxy-4-methyl coumarin, or 4-MU), the detectable label used in the IM.sub.x .RTM. instrument assays (Abbott Laboratories, Abbott Park, Ill.). The substitutions in the 4 position are branched and include functional groups for coupling to biological molecules.
A number of fluorometric labels are known to one of ordinary skill in the art. However, for compatibility reasons, applicants desired a fluorophore label that had electronic properties substantially similar to the 4-MU utilized in the IM.sub.x .RTM. instrument. Otherwise, the label might fluoresce at a wavelength the instrument could not detect absent special filters and the like. A label optimized to the existing instrument was desired. The search began for a coumarin or umbelliferone nucleus that had an activated or activatable tether which could be coupled to a desired molecule. Of course, the tether had to be one which did not substantially alter the electronic properties of the coumarin nucleus.
A tether to a coumarin nucleus had been obtained in the past by a Pechmann condensation to give a 4-methyl group on the coumarin ring [H. V. Pechmann and C. Duisberg, Chem. Ber. 16, 2119 1883)]. Unfortunately, the 4-methyl compound does not provide useful functional groups which can serve to connect the label to a biological molecule.
A reaction very similar to the Pechmann condensation is described in U.S. Pat. No. 4,618,622 (Schlecker, et al.), whereby certain substituents are placed in the 3 or 4 positions of the coumarin ring by condensation of dihydroxybenzene with a .beta.-ketocarboxylate as follows: ##STR1## where R.sub.1 and R.sub.2 represent the added side chains. Although Schlecker describes a fairly broad group of possibilities for the side chains, only hydrogen, methyl and ethyl are enabled as possible R.sub.1 side chains going into the 4 position of the coumarin nucleus (except for the 3,4 tetramethylene ring). None of the enabled compounds bears a functional group which can be used to tether the fluorophore to a biological molecule. The substituents which may be placed at the 3 ring position do not retain the electronic properties of the coumarin required for fluoresence with the IMx instrument.
The effect of 3-position substitution on electronic structure in coumarins is shown in the .sup.13 C NMR spectra compiled by Parmar and Boll [Mag. Res. Chem., 26, 430-433 (1988)]. If R of the product above is H, the .sup.13 C NMR chemical shift of C-3 is 110 ppm; while if R is CH.sub.2 COOEt, C-3 resonates at 115 ppm. This means that the relative electron density on C-3 has decreased upon alkyl substitution, disturbing the electronic structure of the coumarin nucleus.
Kato, et al., (CA91:74247m and J. Chem. Soc., Perkin Trans. 1(2): 525-528 (1979)) describe a method of forming several compounds, including double ring structures, using keten. Compound 7a is allegedly a 7-hydroxycoumarin with a 2-ethoxycarbonylethyl group attached in the 4 position. However, this conclusion is not supported by the NMR data given for this compound in the second column of page 527.
Thus, the previously known condensation methods were not useful because they do not produce 4-substituted materials with activated or activatable functional groups, and because substitutions at the 3-position did not retain the desired electronic properties. A compound having no substituent at the 3-position was desired because of the need for substantially similar electronic properties as 4-MU.